The generation of axon collateral branches is a fundamental aspect of the development of synaptic connectivity between neurons and their targets, and branching/sprouting is also involved in the recovery of the nervous system following injury. The protrusion of axonal filopodia is the first step in the initiation of a collateral branch. Filopodia that become invaded by axonal microtubules subsequently mature into collateral branches. In this proposal we seek to determine the mechanisms of the initiation of axonal filopodia and their maturation into nascent collateral branches. In the first period of support for this project, our studies have unveiled the earliest steps in the cytoskeletal dynamics underlying the formation of axonal filopodia. We demonstrated that axonal filopodia are formed from precursor structures that we term axonal F-actin patches. The competitive renewal application for this project is based on the novel observation of axonal microdomains of phosphoinositide 3-kinase (PI3K) activity that precede the formation of axonal filopodia, which is in turn dependent on PI3K activity. The studies we propose will advance the field by testing a specific hypothesis that merges the earliest signaling and cytoskeletal events underlying branch formation into a coherent model. Using in vitro and in vivo approaches we will test the hypothesis that localized axonal microdomains of PI3K activity orchestrate the formation of axonal F-actin patches through the recruitment and activity of the Rac1 GTPase, the F-actin nucleating system Arp2/3 and septin GTPases, resulting in the coordinated reorganization of the actin and microtubule cytoskeleton giving rise to collateral branches.